1. Field of the Invention
The present invention relates generally to the field of the transport of serum proteins and antibodies mediated by the Fc receptor, FcRn, and further to the effect on serum half life of agents that interact with the Fc receptor in a pH dependent way.
2. Description of Related Art
IgGs constitute the most prevalent immunoglobin class in the serum of man and other mammals and are maintained at remarkably constant levels. Recent studies indicate that the major histocompatibility complex (MHC)-class I related receptor, FcRn, is involved in the homeostasis of serum IgGs (Ghetie et al., 1996; Junghans and Anderson, 1996; Israel et al., 1996). This receptor most likely acts as a salvage receptor, and this would be consistent with its known ability to transcytose IgGs in intact form across the neonatal gut (Wallace and Rees, 1980; Rodewald and Kraehenbuhl, 1984 ) and yolk sac (Roberts et al., 1990; Israel et al., 1995) or placenta (Kristoffersen and Matre, 1996; Simister et al., 1996; Leach et al., 1996). The interaction site of FcRn on mouse IgG1 (mIgG1) has been mapped using site-directed mutagenesis of recombinant Fc-hinge fragments, followed by analysis of these fragments both in viva and in vitro (Kim et al., 1994b; Medesan et al., 1996; 1997). From these studies, I253 (EU numbering (Edelman et al., 1969)), H310, H435 and to a lesser extent, H436 play a central role in this interaction. These amino acids are located at the CH2-CH3 domain interface (Deisenhofer, 1981), and the mapping of the functional site to these residues is consistent with the X-ray crystallographic structure of rat FcRn complexed with rat Fc (Burmeister etal., 1994b).
The FcRn interaction site encompasses three spatially close loops comprised of sequences that are distal in the primary amino acid sequence. The central role of Fc histidines in building this site accounts for the marked pH dependence (binding at pH 6.0, release at pH 7.4) of the Fc-FcRn interaction (Rodewald and Kiaehenbuhl, 1984; Raghavan et al., 1995; Popov et al., 1996), as the pKa of one of the imidazole protons lies in this pH range. I253, H310, H435 and to a lesser degree, H436, are highly conserved in IgGs of both human and rodent IgGs (Kabat et al., 1991). This, taken together with the isolation of a human homolog of FcRn (Story et al., 1994), indicate that the molecular mechanisms involved in IgG homeostasis are common to both mouse and man and this has implications for the modulation of the pharmacokinetics of IgGs for use in therapy.
To date, in studies to identify the FcRn interaction site on Fc, mutations of Fc-hinge fragments have been made that reduce the serum half lives of the corresponding Fc-hinge fragments (Medesan et al., 1997; Kim et al., 1994a). The correlation between serum half life and binding affinity for FcRn is excellent for these mutated Fc-hinge fragments (Kim et al., 1994b; Popov et al., 1996), suggesting that if the affinity of the FcRn-Fc interaction could be increased, whilst still retaining pH dependence, this would result in an Fc fragment with prolonged serum persistence. Production of such a fragment would be a significant advance in the engineering of a new generation of therapeutic IgGs with improved pharmacokinetics such as increased persistence in the circulation. But to date, no such fragments have been produced.
Immunoglobulin Fc domains are also of great interest for purposes of studying the mechanisms of antibody stabilization, catabolism and antibody interactions with further molecules of the immune system. These include, depending on the class of antibody, interactions with complement, and binding to specific receptors on other cells, including macrophages, neutrophils and mast cells. More detailed knowledge of the biology of Fc regions is important in understanding various molecular processes of the immune system, such as phagocytosis, antibody-dependent cell-mediated cytotoxicity and allergic reactions.
The production of a longer-lived Fc fragment that has increased binding to FcRn would be attractive, since such a fragment could be used to tag therapeutic reagents. Chimeric proteins produced in this manner would have the advantage of high in vivo stability which would allow fewer doses of the agent to be used in therapy and possibly even allow lower doses of the agent to be used through its increased persistence in the bloodstream. Unfortunately,methodology for generating proteins, such as antibody fragments, with increased serum persistence has not yet been developed.